Functional and metabolic effects of adaptive glycerol kinase (GLPK) mutants in Escherichia coli

Abstract

Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.

FIGURE 1.

Kinetics of GlpK mutants. A and B, plot the dependence of the initial velocity (v_o) of each GlpK variant on the concentration of ATP. The initial velocities have been normalized to the V_max for each enzyme. The lines trace the fit of the data to the Michaelis-Menten equation. The values for V_max and K_m-ATP derived from this fit are shown in Table 2. C, FBP inhibition. The specific activity of each variant is shown at different concentrations of FBP. Specific activities (SA) were normalized based on the specific activity (SA₀) of each enzyme with no FBP (100%). The lines show how the data were fitted to the equation modeling FBP inhibition (Eq. 1). The V61I mutant is not appreciably inhibited by FBP so its data could not be fit to the equation; however, simulations using different values of K_0.5 suggest that it must be greater than 100 mm. SA₀ and parameters from the fits are shown in Table 2. D, IIA^Glc inhibition. The points show the specific activity (SA) at the indicated concentration of IIA^Glc normalized to the specific activity at 0 IIA^Glc. The lines show the fit to the equation that models IIA^Glc inhibition (Eq. 2). Values for SA₀ and parameters from the fits are shown in Table 2. Symbols for GlpK allozymes: wild type: filled circle, V61L: filled square, D72V: filled triangle, M271I: open circle, Q37P: open square.

FIGURE 2.

Plots of the apparent dependence of relative fitness on (A) FBP affinity (log₁₀ transformed), (B) apparent molecular weight (an indirect measure of the tetramer dissociation constant), (C) intracellular cAMP, and (D) glpK expression. Relative fitness is expressed as the approximate growth rate increase relative to wild type (h⁻¹, × 100), calculated as described under “Experimental Procedures.” No trend line is included in panel (B) because the relationship does not appear to be linear. The FBP affinity of V61L is simply estimated to be >100 mm, but has been plotted as 150 mm ± 50 mm.

FIGURE 3.

Glycerol kinase mutant strains have reduced intracellular cAMP. Intracellular cAMP concentrations in four glycerol kinase mutant strains are significantly reduced compared with wild type (p = 0.0022, two-tailed one-way ANOVA with Dunnett's post-test). Bars represent average of three independent measurements ± S.D.